Borrelia burgdorferi sensu lato, the etiologic agent of Lyme disease, causes a multi-system disorder that may progress into a persistent infection characterized by significant morbidity. The overall goal of this proposal is to identify and characterize virulent strain associated (VSA) outer membrane (OM) proteins, both those that are outer membrane- spanning (Oms) and lipoproteins, that function as protective immunogens. The investigators' long-term goal is to link protective immunity elicited by these proteins to pathogenic mechanisms. Specifically, this study proposes to: (1) Purify and characterize a 28 and 35 kilodalton (kDa) virulent strain associated (VSA) OM proteins of B. burgdorferi that are specific to infectious isolates. The native forms of the 28 and 35 kDa OM proteins will be purified from OM vesicles derived from B. burgdorferi and their amino terminal or internal peptide sequences determined. These sequences will then be used to identify the genes encoding these proteins by screening the B. burgdorferi genomic database; (2) Characterize four virulent strain associated (VSA) antigens of B. burgdorferi identified from a phage lambda expression library. We will determine the extent of differential expression of the VSA antigen in virulent versus avirulent B. burgdorferi and evaluate whether these antigens are either expressed in infected animals or temperature regulated; and (3) Test these OM VSA antigens identified for their ability to confer protective immunity. Recombinant VSA proteins will be overproduced, purified, and used to immunize mice. The native VSA OM 28 and 35 kDa proteins will be incorporated into liposomes and the resulting proteoliposomes used to immunize mice. Immunized animals will then be challenged with infectious B. burgdorferi. Serum specific for protective immunogens will be tested to determine whether these antibodies confer passive protection and whether antibodies mediate adherence inhibition or preferentially kill in vitro cultivated virulent B. burgdorferi in a complement dependent manner. Determination of the protective abilities of different B. burgdorferi antigens expressed in the host relative to those expressed during in vitro cultivation, will facilitate in the development of a vaccine cocktail to protect against non-clonal, environmental isolates of B. burgdorferi. Furthermore, these studies will provide insight into pathogenic mechanisms mediated by VSA antigens of B. burgdorferi.